1. Field of the Invention
The present invention relates to the detection of a DNA sequence in real time using fluorescence.
2. Background Information
The polymerase chain reaction (PCR) developed by Saiki et al. [Science 230, 1350-1354 (1985)] provided a method for rapidly amplifying small specific segments of DNA. The PCR technique has greatly simplified the analysis of DNA sequences at the genomic level.
Detection or identification of a segment of DNA involves a two step procedure. The first step is the PCR amplification reaction using carefully selected primers. The second step identifies whether the desired DNA segment was amplified.
Prior to the present invention, the second step has required the use of a radioactively labeled DNA probe to precisely identify a PCR amplified DNA segment. There are several disadvantages associated with the use of a radioactively labeled probe. For example, radioactive probes are dangerous to handle and when labeled with .sup.32 P, which is the standard protocol, the probe has a short useful life (a few weeks at most). In addition, the use of a radioactive probe prevents detection of the results in real time, that is, a delay for an autoradiographic exposure time is required.